1. Field of the Invention
The invention relates to a multi-purpose reagent system and method for enumeration of red blood cells, white blood cells and thrombocytes and differential determination of white blood cells.
2. Description of the Prior Art
The art recognizes that the ability to resolve the various populations of blood cells into their constituent classes, particularly those of the leukocytes, provides an invaluable diagnostic aid in the study, diagnosis and treatment of various diseases. As is further appreciated, the greater the number of sub-populations that are identifiable and enumeratable, the more accurate and reliable the identification of any single sub-population is likely to be.
Previous scientific publications and patents have described reagents and methods for enumerating these cell types by a variety of mechanisms. U.S. Pat. No. 4,485,175 (to Ledis, et al.) describes a reagent system comprising a multipurpose blood diluent and a lysing reagent, and a method for utilizing same to produce a hemoglobin measurement and differentiation of white blood cells into at least one and up to three sub-populations of leukocytes. U.S. Pat. No. 5,731,206 (to Ledis, et al.) describes a lytic reagent composition, a kit of a lytic reagent system and a method for isolating, identifying and analysis of at least one and up to five sub-populations of leukocytes from a whole blood sample. Other representative patent references pertinent to this field include U.S. Pat. Nos. 3,874,852; 4,286,963; 4,346,018; 4,528,274 and 4,751,179. All are hereby incorporated by reference in their entirety.
This invention concerns certain novel compositions of matter (reagents) and novel methods of use of a set of reagents designed and matched for electronic enumeration and sizing of blood cells, utilizing automated and semi-automated hematology analyzers for the multiple purposes of counting and sizing red blood cells; counting, sizing and differentiating white blood cells according to their subtype; counting and sizing platelets in whole blood; and the photometric measurement of hemoglobin concentration. Effective measurement of the formed elements of a whole blood sample requires multiple steps, including counting and sizing of the red blood cells and platelets, followed by addition of a red blood cell hemolyzing reagent, thus permitting the enumeration and sizing of the remaining white blood cells.
Use of the present invention enhances the ability to resolve the various populations of blood cells into their constituent classes, particularly those of the leukocytes, and provides an invaluable diagnostic aid in the study, diagnosis and treatment of various diseases. As may be further appreciated, the greater the number of sub-populations that are identifiable and enumeratable, the more accurate and reliable the identification of any single sub-population is likely to be.
The diluent reagent of the present invention comprises an aqueous solution of chemical salts providing an electrically conductive solution to which a blood sample can be added to dilute the red blood cells, white blood cells and platelets to enable electronic counting, sizing and evaluation by d.c. impedance, rf conductivity or opacity (a normalization of the rf signal divided by the d.c. signal) and/or light scatter at one or more angles to the incident light beam. In order to separate the complex electrical signals emanating from such a dilute mixture, a first red blood cell lysing reagent is added to rupture (stromatolyse) the red blood cells and dissolve their membrane particles (stroma), such that the red blood cells no longer register in any of the measurements made by the analyzer system. The resulting solution may be counted, sized and evaluated by electronic and mathematical means to produce a volumetric representation of the remaining white blood cells into at least one size population up to three or more size populations. Simultaneously, the lysing reagent also reacts with the hemoglobin liberated from the lysed red blood cells, converts it chemically to a stable oxyhemoglobin derivative, which is then measured photometrically, and a hemoglobin concentration is calculated from a previous known hemoglobin concentration used as a standard.
In a second part of the invention, a lysing reagent is reacted with a blood sample, similarly destroying the red blood cells under acidic conditions of low osmolality and conductivity with an acid active natural detergent present, and resulting in the differentiation of white blood cells (leukocytes) into at least one and up to five component subtypes, specifically described by their morphology and function as lymphocytes, monocytes, granulocytes, eosinophils and basophils. Addition of a third reagent, called a quenching reagent, is required in order to adjust the pH to approximately neutral (i.e., 6.5 to 7.5) and to adjust the conductivity to about 18 to 20 milliSiemens per kilogram, as is usual for isotonically diluted blood specimens. Simultaneous measurement of parameters, such as volume, conductance or opacity, and light scattering ability have been used to differentiate white blood cell subtypes into at least one and up to five different categories. While functional tests of white blood cell subtypes are usually difficult to perform in automated analyzers, these electronic and optical parameters have been related to cellular function by scientific studies designed to correlate cellular function with cell volume, opacity and/or light scatter signals.
Although previous attempts to accomplish the foregoing have permitted performance of similar cellular enumerations and differentiations more or less accurately, nevertheless, the presence of hematological abnormalities and/or aging of the blood specimen post-phlebotomy which cause changes in the cellular response to chemical and electrical stimuli as provided by the analyzer system, have preclused prior compositions and methods from achieving the results of the present invention. These changed cellular responses usually result in changes in the chemical response, and subsequently, the appearance of the histogram and scattergram patterns of the cells. The subtypes are subsequently distorted from the usual presentation of fresh blood cells within the analyzer""s output. This shifting or changing of the cellular response to measurement results in inaccuracies and possible misclassifications that may have medical consequences. These shifts also may cause error flags to be generated by the analyzer system that may further distort the true analytical picture. Additionally, the presence of such error flags requires the laboratorian to re-analyze the specimen by alternative means in order to provide a reliable result to the physician. Such is not the result, however, in the performance of the present invention.
The present invention overcomes and solves these problems encountered in the prior art by treating the abnormal and older, more fragile blood cells in a chemically more gentle manner, from which they can be measured and analyzed closer to their native, whole-blood state. Hematological analysis with better accuracy and fewer error flags is much more desirable in the medical laboratory. Thus, the clinician may receive more useful diagnostic information, eliminating the potential for erroneous data.
The task of improving the reagent designs and the analyzer system performance is made more difficult by the design of current hematology analyzer systems, which usually have fixed counting and sizing thresholds, fixed mathematical treatments (algorithms) of the measured data, and therefore, do not allow much leeway in the reagent chemistry for changes in cellular volumetric size and variant responses to the instrument algorithms. Changes in the analyzer""s positioning of various cell subtypes due to cellular abnormalities and sample-age related changes in chemical behavior generally have resulted in misidentification or misclassification by the analyzer system in the past. By the use of the present invention and the application of a gentler, more cell-friendly chemistry within the analyzer system, cell positioning and electrical response changes can be reduced, thus, presenting a cell count and distribution more nearly like that of fresh blood specimens.
Several different models of electronic blood cell counting and sizing apparatus are useful in practicing the art of blood cell analysis, including Beckman Coulter Models MD, MD II, T Series analyzers (T540, T660, T890, ST, JT 1, JT 2 and JT 3), S Plus 4, 5 and 6, STKR, STKS, MAX-M and Genxe2x80xa2S; and other representative analyzers, such as Abbott Laboratories"" CD3000, CD3500 and others; and TOA (Sysmex) Model 1600 and others.
It is an object of this invention to improve and remedy the many known deficiencies of hematology analysis systems, as discussed above, and additionally to simplify the reagents, remove any toxic components, remove any components that destabilize the hematology analysis system and to increase the stability of the hematology analysis system measurements, while reducing the sensitivity of the analyzer system to the effects of sample age and physiological abnormalities of the patient.
It is an additional object of this invention to provide a stable blood diluent environment for the analysis red blood cells and platelets, both from fresh patient samples and those that have aged since sample collection.
It is a further object of this invention to provide a reagent for a chemical reaction with a whole blood sample, whether freshly collected or aged since collection, that may permit the enumeration of the total number of white blood cells present, the differentiation of at least one and up to three sub-populations of leukocytes, and simultaneously provide a stable hemoglobin chromagen for determination of the hemoglobin concentration by photometric measurement, without requiring the use of toxic compounds such as cyanide to form said stable chromagen.
It is a still further object of this invention to provide an additional reagent system that can further subdivide leukocyte sub-populations of freshly collected or aged blood specimens into at least one and up to five distinct classifications for use in differential determination of the relative number of such cells in each sub-population by providing a means to lyse the red blood cells, then to stabilize the residual white blood cell mixture by addition of a quenching reagent for subsequent analysis.
It is yet another additional object of this invention to provide a novel reagent system for effective use in separating and differentiating white blood cell subpopulations, whether freshly collected or aged since collection, for the purpose of measurement by purely physical, electrical and light scattering means, and by immunological and immunochemical means by use of additional reagents comprising antibodies or antisera specific for one or more marker molecules on the surface or within the cellular contents of said sub-populations.
It is a further object of this invention to provide a method for the performance of these analyses, including: a) the counting and sizing of red blood cells and platelets from a fresh or aged blood sample, b) the removal of red blood cells and the subsequent measurement of at least one up to three sub-populations of white blood cells from freshly collected or aged blood samples, c) the conversion of the liberated hemoglobin to a stable derivative for photometric measurement, d) the differential determination of at least one and up to five sub-populations of leukocytes from freshly collected or aged blood samples by measurement of physical, electrical and light scatter properties, and e) measurement of the immunochemical or immunological response of any such sub-populations to antibodies or antiserum specific for one or more marker molecules on the surface or within the cellular contents of said sub-populations.
Other and further objects and advantages of the present invention will become more apparent from the detailed description of the preferred embodiments as set forth in the following when taken together with the drawings.